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Phenoloxidase activity in larval and juvenile homogenates and adult plasma and haemocytes of bivalve molluscs
Fish & Shellfish Immunology Fish & Shellfish Immunology 15 (2003) 275–282 www.elsevier.com/locate/fsi
Phenoloxidase activity in larval and juvenile homogenates and adult plasma and haemocytes of bivalve molluscs Antonio Luna-Gonza´lez a, Alfonso N. Maeda-Martı´nez a,*, Francisco Vargas-Albores b, Felipe Ascencio-Valle a, Miguel Robles-Mungaray a b
a Centro de Investigaciones Biolo´gicas del Noroeste (CIBNOR), P.O. Box 128, La Paz, B.C.S. 23090, Mexico Centro de Investigacio´n en Alimentacio´n y Desarrollo (CIAD), P.O. Box 1735, Hermosillo, Sonora 83000, Mexico
Received 25 July 2002; accepted 11 November 2002
Abstract Phenoloxidase (PO) activity was studied in larval and juvenile homogenates and in the plasma and haemocytes of adult Crassostrea gigas, Argopecten ventricosus, Nodipecten subnodosus, and Atrina maura. Samples were tested for the presence of PO activity by incubation with the substrate -3, 4-dihydroxyphenylalanine using trypsin, -chymotrypsin, laminarin, lipopolysaccharides (LPS), and sodium dodecyl sulphate (SDS) to elicit activation of prophenoloxidase (proPO) system. PO activity was not detected in larval homogenate. In juvenile homogenate, PO activity was found only in C. gigas and N. subnodosus. PO activity was present in adult samples and was enhanced by elicitors in the plasma of all species tested, but in haemocyte lysate supernatant (HLS) of only N. subnodosus. Activation of proPO by laminarin was suppressed by a protease inhibitor cocktail (P-2714) in plasma and HLS of all species tested. 2003 Elsevier Ltd. All rights reserved. Keywords: Crassostrea gigas; Argopecten ventricosus; Nodipecten subnodosus; Atrina maura; Prophenoloxidase; Haemocytes; Immunology; Larvae; Juveniles
1. Introduction There are at least two phenoloxidase (PO) types: o-diphenoloxidase (E.C. 1.10.3.1) and tyrosinase (E.C. 1.14.18.1). PO is a copper-dependent enzyme capable of mediating the conversion of phenols such as -3, 4-dihydroxyphenylalanine (-DOPA), to unstable quinones, which afterwards are transformed to melanin following a non-enzymatic pathway [1–3]. This enzyme appears to be widespread in microorganisms, plants, and animals [4–6] and is the major enzyme produced when the prophenoloxidase (proPO) system is activated by -1, 3-glucans [7–12], bacteria cell walls [7,13], and lipopolysaccharides (LPS) from Gram-negative bacteria [12,14–16]. In arthropods, the proPO system is known to be part of the internal defence system [15,17]. The activation process of changing proPO to PO involves the action of a serine * Corresponding author. Fax: +52-612-123-2753. E-mail address: [email protected] (A.N. Maeda-Martı´nez). 1050-4648/03/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1050-4648(02)00165-1
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protease called proPO-activating enzyme (PPAE) [18–20]. Chemicals such as SDS, urea, and ethanol have the ability to activate the proPO system [9,11,21], probably by causing structural changes in the protein molecule, rather than proteolytic cleavage [11]. The proPO system and its associated proteins seem to be involved in non-self recognition and cellular communication in arthropods [17,19,22]. However, there are only a few reports on the presence and localization of proPO in molluscs, on the possible activating system, or on the nature of this enzyme in defence mechanisms [3]. PO activity has been found in several species of bivalve molluscs [3,12,15,23–28]. However, it was not found in Tridacna crocea [29] or Perna perna [30]. The presence of PO in the haemolymph of molluscs and stimulation of enzyme activity by bacterial and fungal cell wall components have confirmed that PO plays a role in an internal defence mechanism [28]. To date, only the work of Asokan et al. [12] has demonstrated proPO system activation in the mussel Perna viridis by bacterial and fungal cell wall components, and the activation of proPO to PO by a serine protease suggesting that proPO system in P. viridis serves an important function in non-self recognition and host immune reactions. In bivalves, susceptibility to bacterial infections is higher in larvae than in adults [31]. This produces high economic losses in hatcheries and uncertainty of the amount of spat to be obtained [32–35]. Apart from good practices and addition of antibiotics to the cultures, few measures can be taken to prevent larval mortality. Knowledge on the internal defence mechanisms in early stages of bivalves could offer alternatives, but unfortunately, this is rare. Most immunological studies have been done in adult forms, probably because it is not possible to get blood samples from larvae and young juveniles to allow the study of the haemocytes responsible for the internal defence. In Crassostrea virginica, Elston [36,37] studied the larval haemocytes (coelomocytes) and classified these cells into phagocytic and non-phagocytic free-living cells, whereas in Mytilus edulis, the presence of some immunological elements commonly found in adults, such as PO, arylsulphatase, phagocytosis, and reactive oxygen metabolites, were demonstrated in trochophore and veliger larvae [24]. In Mytilus galloprovincialis, Mitta et al. [38] recently found the antimicrobial peptide mytilin, during larval metamorphosis, and defensin, after metamorphosis is accomplished. In the present work, we examined in vitro PO activity and the activation of the proPo system by several elicitors in larval and juvenile homogenates of the Pacific oyster Crassostrea gigas, the catarina scallop Argopecten ventricosus, and the lion paw scallop Nodipecten subnodosus, and in haemolymph of adults of these three species and the penshell Atrina maura. 2. Materials and methods 2.1. Larvae Adult C. gigas, A. ventricosus, and N. subnodosus were brought to the hatchery at CIBNOR and were conditioned for spawning for at least 15 days in 1100-l fibreglass tanks containing aerated seawater at 241 (C and 36‰. Broodstock were fed 1.5105 cells ml1 of an algal mixture of Chaetoceros calcitrans, Isochrysis galbana, and Chaetoceros gracilis (1:1:2). Half of the water was changed daily. After the conditioning period, the bivalves were induced to spawn by thermal shock (one species at a time) and the resulting larvae were cultured in 5000-l conical tanks at a density of 5–7 larvae ml1, with filtered (1 µm) seawater at 241 (C, 36‰. Total water change was done every 3 days. The larvae were fed 3.0105 cells ml1 of I. galbana and C. calcitrans (1:1). 2.2. Preparation of larval homogenate supernatant (LHS) At day 6, larvae of C. gigas (3.5106; shell length, 115.24 µm), A. ventricosus (3.5106; shell length, 128.316 µm), and N. subnodosus (3.5106; shell length, 117.416 µm) were placed in separate 400-l conical fibreglass tanks for 24 h at a density of 4 larvae ml1, with filtered seawater (1 µm) at 241 (C,
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36‰. Larvae were not fed in order to empty their stomachs. At day 7, larvae were washed three times with filtered sterile seawater, and resuspended in 10 ml pre-cooled Tris–HCl buffer (250 mM Tris; pH, 6.5). Ten 1 ml samples were placed in pre-cooled Eppendorf tubes and homogenized with a Kontes pellet pestle motor. The larval homogenate was centrifuged at 48,000g for 30 min at 4 (C in an ultracentrifuge (Model L7, Beckman) and the clear supernatant (LHS) was pooled and used immediately in all assays. 2.3. Juveniles Larvae of C. gigas, A. ventricosus, and N. subnodosus were obtained as previously described and cultured in the hatchery. Juveniles were maintained in the hatchery in 1200-l fibreglass tanks with an up-welling system and continuous seawater flow at 241 (C and 36‰. Juveniles were fed 1.5105 cells ml1 of an algal mixture of C. calcitrans, I. galbana, and C. gracilis (1:1:2). 2.4. Preparation of juvenile homogenate supernatant (JHS) Juveniles of C. gigas (9.01.6 mm shell length, 170 days old), A. ventricosus (8.61.0 mm shell length, 160 days old), and N. subnodosus (4.30.6 mm shell length, 70 days old) were used to obtain the JHS. Flesh was removed from valves of 15 specimens, each of A. ventricosus and C. gigas, washed with filtered sterile seawater, and put in 2.5 ml of pre-cooled 250 mM Tris–HCl buffer at pH 6.5. Eighty specimens of N. subnodosus were washed with filtered, sterile seawater without removing flesh from the valves. Samples were homogenized with a Kontes pellet pestle motor in pre-cooled Eppendorf tubes. The juvenile homogenate was then centrifuged at 48,000g for 30 min at 4 (C in an ultracentrifuge (Model L7, Beckman), and the clear supernatant (JHS) was pooled and used immediately in all assays. 2.5. Adults Adult C. gigas (shell length, 123.316.9 mm), A. ventricosus (shell length, 57.42.5 mm), N. subnodosus (shell length, 134.210.8 mm), and A. maura (shell length, 184.413.8 mm) were collected from culture sites in Bahı´a Magdalena and Laguna San Ignacio, B.C.S., Mexico. In the laboratory, specimens were placed in 1100-l fibreglass tanks containing seawater at 241.0 (C and 36‰. They were fed 1.5105 cells ml1 of an algal mixture of C. calcitrans, I. galbana, and C. gracilis (1:1:2). Half of the water was changed daily. 2.6. Haemolymph collection In a previous study, we found that the haemolymph of the molluscs does not coagulate and haemocytes remain free and refractile without any sign of degranulation for at least 30 min. Therefore, an anticoagulant was not necessary, as in P. viridis [12]. A. ventricosus and N. subnodosus were bled 1.0 ml from the posterior adductor muscle by inserting a 27-gauge needle attached to a 1-ml sterile plastic syringe. C. gigas and A. maura were bled 1.0 ml by cardiac puncture with the same kind of syringe and needle. The samples collected from 30 specimens of each species were pooled in sterile glass tubes held on ice. 2.7. Separation of plasma and haemocytes Haemolymph samples from each species were immediately centrifuged at 400g for 5 min at 4 (C, and the resulting supernatants were used as plasma. The pellet composed of the haemocytes was resuspended and was washed once with Tris-buffered saline (50 mM Tris; 400 mM NaCl, pH 7.5). The haemocytes were finally suspended in 5 ml 250 mM Tris–HCl buffer at pH 6.5 [12].
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2.8. Preparation of haemocyte lysate supernatant (HLS) The haemocyte suspension was homogenized (20 kHz/100 W, 430 s, 4 (C) in an ultrasonic homogenizer (Cole Parmer). The resulting cell homogenate was then centrifuged at 48,000g for 30 min at 4 (C in an ultracentrifuge (Model L7, Beckman). The clear supernatant, representing HLS, was used in all assays. 2.9. Assays for the activation of proPO PO activity was measured four times (each one in quadruplicate) spectrophotometrically by recording the formation of dopachrome from -DOPA (Sigma), according to Asokan et al. [12]. Enzyme activity for all assays was expressed as units, where one unit represents the change in absorbance min1 mg protein1[35]. Previous studies were made for each species to determine the highest rate of PO activity after the incubation of the reaction mixtures with -DOPA. Times of incubation were 5, 10, 20, 30, and 60 min. In all assays, the highest rate of PO activity was obtained consistently at 5 min, and the activity remained at essentially the same level till 60 min; therefore, absorbance was recorded after 5 min. Value less than 2 units was considered negative [15]. Trypsin-activated HLS from Penaeus californiensis [11] was used as the proPO positive control. 2.10. Effect of trypsin, -chymotrypsin, laminarin, LPS, and SDS as elicitors All elicitors were purchased from Sigma. Trypsin (2 mg ml1), laminarin from Laminaria digitata (3 mg ml1), -chymotrypsin (2 mg ml1), LPS from Escherichia coli 055:B5 (3 µg ml1), and SDS (6 mg ml1) were diluted in pre-cooled Tris–HCl buffer (250 mM Tris; pH, 6.5). LHS (50 µl), JHS, plasma, or HLS samples were placed on a micro-well plate and incubated with 50 µl of the elicitor. Thereafter, 50 µl of 5 mM -DOPA was added as substrate. Optical density at 490 nm was recorded at 5, 10, 20, 30, and 60 min, using a micro-plate reader (Model 350, Bio–Rad). For the control, 50 µl of a sample was mixed with 50 µl of buffer and 50 µl of substrate. As blanks, 100 µl buffer was mixed with either 50 µl -DOPA to detect possible spontaneous oxidation of this substrate by light, or 50 µl of a sample to eliminate sample colour. 2.11. Effect of a protease inhibitor on proPO activation The effect of protease inhibitor cocktail P-2714 (Sigma, St Louis, MO) on laminarin-induced proPO activation in plasma and HLS in C. gigas, A. ventricosus, N. subnodosus, and A. maura was studied. In this experiment, 50 µl JHS, plasma, or HLS samples were pre-incubated with 25 µl of P-2714 at room temperature for 15 min. After that, 25 µl of 3 mg ml1 laminarin was added and further incubated at room temperature for 15 min. Three different controls were run simultaneously during the course of this experiment: JHS, plasma, or HLS samples were pre-incubated with Tris–HCl buffer as buffer control, laminarin, as positive control or P-2714 as negative control. Each 100-µl reaction volume was incubated with 50 µl of -DOPA, and PO activity was recorded. The amount of measurable proPO for laminarin, determined by subtracting PO activity in the buffer control from that of the positive control, was considered as 100%, and the level of proPO that remained in JHS, plasma, or HLS sample, after reaction with inhibitor and laminarin, was expressed as percent residual activity. 2.12. Protein determination Protein concentration of the studied samples was determined using the Bradford method [39], with bovine serum albumin (BSA) from Sigma as standard. The protein concentrations of mollusc samples analyzed were usually in the range of 0.12–3.50 mg ml1. Protein concentration in plasma and HLS of shrimp was 43.42 and 1.42 mg ml–1, respectively.
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Table 1 PO activity (units min1 mg protein1103) in plasma and haemocyte lysate of adults of four bivalve species, activated with trypsin, -chymotrypsin, SDS, laminarin, and LPS, and the effect of protease inhibitor cocktail P-2714 on proPO activation by laminarin Treatment
Control Trypsin -Chymotrypsin SDS Laminarin LPS P-2714 + Laminarin
Crassostrea gigas
Argopecten ventricosus
Nodipecten subnodosus
Atrina maura
HLS
Plasma
HLS
Plasma
HLS
Plasma
HLS
Plasma
4.52.3 9.82.7 9.33.9 14.62.3 9.63.8 8.60.5 52
7.35.4 18.7.0.5* 19.00.9* 22.12.9** 20.30.5** 19.20.5* 43
2.70.37 5.61.2 4.71.5 5.21.3 5.61.8 4.10.4 53
25.54.8 67.23.5** 55.52.5** 54.45.3** 50.57.9* 32.87.9 59
2.80.4 6.10.2** 4.40.5* 5.20.3** 7.00.6** 5.70.8** 54
18.50.6 32.23.2** 37.10.6** 40.72.2** 44.21.8** 42.82.2** 67
4.60.6 15.93.9 16.24.2 18.55.3 19.14.6 8.61.4 58
14.82.6 31.46.0* 25.93.7 27.43.7 33.73.9* 16.64.1 51
Values are the mean of four determinations, each one in quadruplicateSD. Values of inhibition by P-2714 are the mean of four determinations, each one in quadruplicate, and expressed as percent residual activity. * and ** denote significant differences as compared to control at P<0.05 and P<0.001, respectively.
2.13. Statistical analysis A one-way analysis of variance (ANOVA) using the F test was applied to analyse the differences among treatments and control and among species. P values lower than 0.05 were used to identify significant differences. Where significant ANOVA differences occurred among averages, Tukey’s HSD test was used to identify the nature of these differences at P<0.05. 3. Results 3.1. PO activity in larvae Control values for C. gigas, A. ventricosus, and N. subnodosus were 0.80.09, 0.70.13, 1.20.21, respectively. There was no PO activity either in the buffer control or after incubation of samples with proteolytic elicitors trypsin and -chymotrypsin, anionic detergent SDS, and microbial molecules laminarin and LPS. Large amounts of protein (2.18–3.5 mg ml1) were found in larval pools. 3.2. PO activity in juveniles PO activity was detected only in JHS of C. gigas and N. subnodosus. The activity obtained with the elicitors was not significantly different from the buffer control (3.90.59, 1.90.55, respectively). As in larvae, relatively large amounts of protein (0.88–1.06 mg ml1) were found in juvenile pool. 3.3. PO activity in HLS and plasma of adults Oxidation of -DOPA occurred in the buffer control of the four bivalve species (Table 1 ). Significant differences in the PO activity between buffer control and elicitors occurred in the plasma of all species, but in HLS of only N. subnodosus. The amounts of protein in HLS (0.23–1.4 mg ml1) and plasma (0.12–0.22 mg ml1) were lower than in larval and juvenile homogenates. 3.4. Effect of a protease inhibitor on proPO activation In the present study, the effect of P-2714 on the activation of proPO by laminarin was examined in the samples of C. gigas, A. ventricosus, N. subnodosus, and A. maura. Important reduction of PO activity was
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observed in HLS and plasma. The incubation of samples with P-2714, before reaction with 3 mg ml1 laminarin, consistently reduced PO activity up to 57% (Table 1). 4. Discussion In this study, PO activity was not detected in larval homogenates of C. gigas, A. ventricosus, and N. subnodosus. However, Dyrynda et al. [24] found PO activity in all disaggregated larval (trochophore and veliger) cells from M. edulis, using a cytochemical staining method. This discrepancy may be a consequence of the method used by Dyrynda et al. [24] and that employed here. In this study, we demonstrated that our method is reliable, since PO activity was found in two species of juveniles, which indicates the good resolution of the technique employed. Moreover, homogenates gave good results for determining lysozyme-like activity and other lysosomal enzymes in ripe eggs, larvae at different stages, and juveniles of C. gigas (Luna-Gonza´lez et al., unpublished). If the mollusc larvae lack a proPO system, these organisms, at this stage, would lack an important element of the defence system found in adults, partially explaining the high susceptibility of the bivalve larvae to bacterial infections [32–34,40–44]. Although detection of PO activity was positive in juvenile C. gigas and N. subnodosus, elicitors did not enhance PO activity and, therefore, the role of PO in juveniles of these species remains unclear. The lack of PO in A. ventricosus cannot be explained. Further work is needed to demonstrate the participation of other components of the defence system of adults in larvae, such as blood cells, antimicrobial proteins, lectins, lysosomal enzymes, superoxide anion, and peroxynitrite. Also to be explained is why PO was not activated by the elicitors tested, as it occurs in adults, and why PO activity was not detected in A. ventricosus juveniles. In adult molluscs, it is known that PO has an important role in shell growth and repair [23,45] and as part of the defence system [12]. In the present study, the spectrophotometric analysis revealed the oxidation of -DOPA by both HLS and plasma of adult C. gigas, A. ventricosus, N. subnodosus, and A. maura. Incubation of samples with elicitors enhanced PO activity in the samples of all species tested, suggesting the presence of a proPO system and its possible role in defence reactions, as was demonstrated by Asokan et al. [12] for the mussel P. viridis. In this mussel, PO activity was induced by trypsin as well as LPS and -1, 3-glucans. In other bivalve species, such as M. edulis, pre-incubation of the cells with 0.025% zymosansupernatant produced a ten-fold increase in the PO activity [3]. ProPO activity was also induced by trypsin in the gastropod mollusc Biomphalaria glabrata [46]. In many insects, crustaceans, and the bivalve P. viridis, the activation of proPO by non-self molecules has been shown to be susceptible to inhibition by serine protease inhibitors [9,12,47,48]. In this work, the incubation of plasma and HLS with protease inhibitor cocktail P-2714, prior to reaction with laminarin, prevented proPO activation up to 57%. The present study establishes that PO is present in juvenile C. gigas and N. subnodosus and its activity can be enhanced by the elicitors tested in adult specimens of the four species studied, suggesting that the proPO system plays a role as a defence mechanism.
Acknowledgements The authors are grateful to Cultivos del Mar Sudcaliforniano S. A. de C. V. and to Sol Azul S. A. de C. V. for providing broodstock and experimental organisms employed in this study. This work was supported by Consejo Nacional de Ciencia y Tecnologı´a, Mexico (CONACyT, project G-33953-B) and a PhD-student grant to A. Luna, and by CIBNOR project PAC-5. Thanks to Dr Jorge Herna´ndez Lo´pez for his critical review and comments on a previous version of this manuscript. CIBNOR editing staff improved the English language text.
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[30] Barracco MN, Dias I, Moreira FM. Some haemato-immunological parameters in the mussel Perna perna. Fish Shellfish Immunol 1999;9:387–404. [31] Lane E, Birkbeck TH. Species specificity of some bacterial pathogens of bivalve molluscs is correlated with their interaction with bivalve haemocytes. J Fish Dis 2000;23:275–9. [32] Tubiash HS, Chanley PE, Leipson E. Bacillar necrosis a disease of larval bivalve mollusks. 1. Etiology and epizootiology. J Bacteriol 1965;90:1036–44. [33] Elston RA. Prevention and management of infectious diseases in intensive mollusc husbandry. J World Maricult Soc 1984; 15:284–300. [34] Sugumar G, Nakai T, Hirata Y, Matsubara D, Muroga K. Vibrio splendidus biovar II as the causative agent of bacillary necrosis of Japanese oyster Crassostrea gigas larvae. Dis Aquat Organ 1998;33:111–8. [35] Vargas-Albores F, Barracco MA. Mecanismos de defensa de los moluscos bivalvos, con e´nfasis en pectı´nidos. In: Maeda-Martı´nez AN, editor. Los moluscos Pectı´nidos de Iberoame´rica: Ciencia y Acuicultura. Me´xico: Editorial Limusa; 2001, pp. 127–46. [36] Elston RA. Functional anatomy, histology and ultrastructure of the soft tissues of the larval American oyster, Crassostrea virginica. Proc Natl Shellfish Assoc 1980;70:65–93. [37] Elston RA. Functional morphology of the coelomocytes of the larval oysters Crassostrea virginica and Crassostrea gigas. J Mar Biol Assoc UK 1980;60:947–57. [38] Mitta G, Vandenbulcke F, Hubert F, Salzet M, Roch P. Involvement of mytilins in mussel antimicrobial defense. J Biol Chem 2000;275:12954–62. [39] Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–54. [40] DiSalvo LH, Blecka J, Zebal R. Vibrio anguillarum and larval mortality in a California coastal shellfish hatchery. Appl Environ Microbiol 1978;35:219–21. [41] Nicolas JL, Ansquer D, Cochard JC. Isolation and characterization of a pathogenic bacterium specific to Manila clam Tapes philippinarum larvae. Dis Aquat Organ 1992;14:153–9. [42] Nicolas JL, Corre S, Gauthier G, Robert R, Ansquer D. Bacterial problems associated with scallop (Pecten maximus) larval culture. Dis Aquat Organ 1996;27:67–76. [43] Lambert C, Nicolas JL, Cilia V, Corres S. Vibrio pectenicida sp. Nov., a pathogen of scallop (Pecten maximus) larvae. Int J Syst Bacteriol 1998;48:481–7. [44] Sainz JC, Maeda-Martı´nez AN, Ascencio F. Experimental vibriosis induction with Vibrio alginolyticus of larvae of the cataria scallop (Argopecten ventricosus=circularis) (Sowerby II, 1842). Microb Ecol 1999;35:188–92. [45] Jones GM, Saleuddin ASM. Cellular mechanisms of periostracum formation in Physa spp. (Mollusca: Pulmonata). Can J Zool 1978;56:2299–311. [46] De Aragao GA, Bacila M. Purificacion and properties of a polyphenoloxidase from the fresh water snail, Biomphalaria glabrata. Comp Biochem Physiol 1976;54:179–82. [47] Lanz H, Herna´ndez S, Garrido-Guerrero E, Tsutsumi V, Are´chiga H. Prophenoloxidase system activation in the crayfish Procambarus clarki. Dev Comp Immunol 1993;17:399–406. [48] Aspa´n A, So¨derha¨ll K. Purification of prophenoloxidase from crayfish blood cells, and its activation by an endogenous serine proteinase. Insect Biochem 1991;21:363–73.
Phenoloxidase activity in larval and juvenile homogenates and adult plasma and haemocytes of bivalve molluscs Antonio Luna-Gonza´lez a, Alfonso N. Maeda-Martı´nez a,*, Francisco Vargas-Albores b, Felipe Ascencio-Valle a, Miguel Robles-Mungaray a b
a Centro de Investigaciones Biolo´gicas del Noroeste (CIBNOR), P.O. Box 128, La Paz, B.C.S. 23090, Mexico Centro de Investigacio´n en Alimentacio´n y Desarrollo (CIAD), P.O. Box 1735, Hermosillo, Sonora 83000, Mexico
Received 25 July 2002; accepted 11 November 2002
Abstract Phenoloxidase (PO) activity was studied in larval and juvenile homogenates and in the plasma and haemocytes of adult Crassostrea gigas, Argopecten ventricosus, Nodipecten subnodosus, and Atrina maura. Samples were tested for the presence of PO activity by incubation with the substrate -3, 4-dihydroxyphenylalanine using trypsin, -chymotrypsin, laminarin, lipopolysaccharides (LPS), and sodium dodecyl sulphate (SDS) to elicit activation of prophenoloxidase (proPO) system. PO activity was not detected in larval homogenate. In juvenile homogenate, PO activity was found only in C. gigas and N. subnodosus. PO activity was present in adult samples and was enhanced by elicitors in the plasma of all species tested, but in haemocyte lysate supernatant (HLS) of only N. subnodosus. Activation of proPO by laminarin was suppressed by a protease inhibitor cocktail (P-2714) in plasma and HLS of all species tested. 2003 Elsevier Ltd. All rights reserved. Keywords: Crassostrea gigas; Argopecten ventricosus; Nodipecten subnodosus; Atrina maura; Prophenoloxidase; Haemocytes; Immunology; Larvae; Juveniles
1. Introduction There are at least two phenoloxidase (PO) types: o-diphenoloxidase (E.C. 1.10.3.1) and tyrosinase (E.C. 1.14.18.1). PO is a copper-dependent enzyme capable of mediating the conversion of phenols such as -3, 4-dihydroxyphenylalanine (-DOPA), to unstable quinones, which afterwards are transformed to melanin following a non-enzymatic pathway [1–3]. This enzyme appears to be widespread in microorganisms, plants, and animals [4–6] and is the major enzyme produced when the prophenoloxidase (proPO) system is activated by -1, 3-glucans [7–12], bacteria cell walls [7,13], and lipopolysaccharides (LPS) from Gram-negative bacteria [12,14–16]. In arthropods, the proPO system is known to be part of the internal defence system [15,17]. The activation process of changing proPO to PO involves the action of a serine * Corresponding author. Fax: +52-612-123-2753. E-mail address: [email protected] (A.N. Maeda-Martı´nez). 1050-4648/03/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S1050-4648(02)00165-1
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protease called proPO-activating enzyme (PPAE) [18–20]. Chemicals such as SDS, urea, and ethanol have the ability to activate the proPO system [9,11,21], probably by causing structural changes in the protein molecule, rather than proteolytic cleavage [11]. The proPO system and its associated proteins seem to be involved in non-self recognition and cellular communication in arthropods [17,19,22]. However, there are only a few reports on the presence and localization of proPO in molluscs, on the possible activating system, or on the nature of this enzyme in defence mechanisms [3]. PO activity has been found in several species of bivalve molluscs [3,12,15,23–28]. However, it was not found in Tridacna crocea [29] or Perna perna [30]. The presence of PO in the haemolymph of molluscs and stimulation of enzyme activity by bacterial and fungal cell wall components have confirmed that PO plays a role in an internal defence mechanism [28]. To date, only the work of Asokan et al. [12] has demonstrated proPO system activation in the mussel Perna viridis by bacterial and fungal cell wall components, and the activation of proPO to PO by a serine protease suggesting that proPO system in P. viridis serves an important function in non-self recognition and host immune reactions. In bivalves, susceptibility to bacterial infections is higher in larvae than in adults [31]. This produces high economic losses in hatcheries and uncertainty of the amount of spat to be obtained [32–35]. Apart from good practices and addition of antibiotics to the cultures, few measures can be taken to prevent larval mortality. Knowledge on the internal defence mechanisms in early stages of bivalves could offer alternatives, but unfortunately, this is rare. Most immunological studies have been done in adult forms, probably because it is not possible to get blood samples from larvae and young juveniles to allow the study of the haemocytes responsible for the internal defence. In Crassostrea virginica, Elston [36,37] studied the larval haemocytes (coelomocytes) and classified these cells into phagocytic and non-phagocytic free-living cells, whereas in Mytilus edulis, the presence of some immunological elements commonly found in adults, such as PO, arylsulphatase, phagocytosis, and reactive oxygen metabolites, were demonstrated in trochophore and veliger larvae [24]. In Mytilus galloprovincialis, Mitta et al. [38] recently found the antimicrobial peptide mytilin, during larval metamorphosis, and defensin, after metamorphosis is accomplished. In the present work, we examined in vitro PO activity and the activation of the proPo system by several elicitors in larval and juvenile homogenates of the Pacific oyster Crassostrea gigas, the catarina scallop Argopecten ventricosus, and the lion paw scallop Nodipecten subnodosus, and in haemolymph of adults of these three species and the penshell Atrina maura. 2. Materials and methods 2.1. Larvae Adult C. gigas, A. ventricosus, and N. subnodosus were brought to the hatchery at CIBNOR and were conditioned for spawning for at least 15 days in 1100-l fibreglass tanks containing aerated seawater at 241 (C and 36‰. Broodstock were fed 1.5105 cells ml1 of an algal mixture of Chaetoceros calcitrans, Isochrysis galbana, and Chaetoceros gracilis (1:1:2). Half of the water was changed daily. After the conditioning period, the bivalves were induced to spawn by thermal shock (one species at a time) and the resulting larvae were cultured in 5000-l conical tanks at a density of 5–7 larvae ml1, with filtered (1 µm) seawater at 241 (C, 36‰. Total water change was done every 3 days. The larvae were fed 3.0105 cells ml1 of I. galbana and C. calcitrans (1:1). 2.2. Preparation of larval homogenate supernatant (LHS) At day 6, larvae of C. gigas (3.5106; shell length, 115.24 µm), A. ventricosus (3.5106; shell length, 128.316 µm), and N. subnodosus (3.5106; shell length, 117.416 µm) were placed in separate 400-l conical fibreglass tanks for 24 h at a density of 4 larvae ml1, with filtered seawater (1 µm) at 241 (C,
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36‰. Larvae were not fed in order to empty their stomachs. At day 7, larvae were washed three times with filtered sterile seawater, and resuspended in 10 ml pre-cooled Tris–HCl buffer (250 mM Tris; pH, 6.5). Ten 1 ml samples were placed in pre-cooled Eppendorf tubes and homogenized with a Kontes pellet pestle motor. The larval homogenate was centrifuged at 48,000g for 30 min at 4 (C in an ultracentrifuge (Model L7, Beckman) and the clear supernatant (LHS) was pooled and used immediately in all assays. 2.3. Juveniles Larvae of C. gigas, A. ventricosus, and N. subnodosus were obtained as previously described and cultured in the hatchery. Juveniles were maintained in the hatchery in 1200-l fibreglass tanks with an up-welling system and continuous seawater flow at 241 (C and 36‰. Juveniles were fed 1.5105 cells ml1 of an algal mixture of C. calcitrans, I. galbana, and C. gracilis (1:1:2). 2.4. Preparation of juvenile homogenate supernatant (JHS) Juveniles of C. gigas (9.01.6 mm shell length, 170 days old), A. ventricosus (8.61.0 mm shell length, 160 days old), and N. subnodosus (4.30.6 mm shell length, 70 days old) were used to obtain the JHS. Flesh was removed from valves of 15 specimens, each of A. ventricosus and C. gigas, washed with filtered sterile seawater, and put in 2.5 ml of pre-cooled 250 mM Tris–HCl buffer at pH 6.5. Eighty specimens of N. subnodosus were washed with filtered, sterile seawater without removing flesh from the valves. Samples were homogenized with a Kontes pellet pestle motor in pre-cooled Eppendorf tubes. The juvenile homogenate was then centrifuged at 48,000g for 30 min at 4 (C in an ultracentrifuge (Model L7, Beckman), and the clear supernatant (JHS) was pooled and used immediately in all assays. 2.5. Adults Adult C. gigas (shell length, 123.316.9 mm), A. ventricosus (shell length, 57.42.5 mm), N. subnodosus (shell length, 134.210.8 mm), and A. maura (shell length, 184.413.8 mm) were collected from culture sites in Bahı´a Magdalena and Laguna San Ignacio, B.C.S., Mexico. In the laboratory, specimens were placed in 1100-l fibreglass tanks containing seawater at 241.0 (C and 36‰. They were fed 1.5105 cells ml1 of an algal mixture of C. calcitrans, I. galbana, and C. gracilis (1:1:2). Half of the water was changed daily. 2.6. Haemolymph collection In a previous study, we found that the haemolymph of the molluscs does not coagulate and haemocytes remain free and refractile without any sign of degranulation for at least 30 min. Therefore, an anticoagulant was not necessary, as in P. viridis [12]. A. ventricosus and N. subnodosus were bled 1.0 ml from the posterior adductor muscle by inserting a 27-gauge needle attached to a 1-ml sterile plastic syringe. C. gigas and A. maura were bled 1.0 ml by cardiac puncture with the same kind of syringe and needle. The samples collected from 30 specimens of each species were pooled in sterile glass tubes held on ice. 2.7. Separation of plasma and haemocytes Haemolymph samples from each species were immediately centrifuged at 400g for 5 min at 4 (C, and the resulting supernatants were used as plasma. The pellet composed of the haemocytes was resuspended and was washed once with Tris-buffered saline (50 mM Tris; 400 mM NaCl, pH 7.5). The haemocytes were finally suspended in 5 ml 250 mM Tris–HCl buffer at pH 6.5 [12].
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2.8. Preparation of haemocyte lysate supernatant (HLS) The haemocyte suspension was homogenized (20 kHz/100 W, 430 s, 4 (C) in an ultrasonic homogenizer (Cole Parmer). The resulting cell homogenate was then centrifuged at 48,000g for 30 min at 4 (C in an ultracentrifuge (Model L7, Beckman). The clear supernatant, representing HLS, was used in all assays. 2.9. Assays for the activation of proPO PO activity was measured four times (each one in quadruplicate) spectrophotometrically by recording the formation of dopachrome from -DOPA (Sigma), according to Asokan et al. [12]. Enzyme activity for all assays was expressed as units, where one unit represents the change in absorbance min1 mg protein1[35]. Previous studies were made for each species to determine the highest rate of PO activity after the incubation of the reaction mixtures with -DOPA. Times of incubation were 5, 10, 20, 30, and 60 min. In all assays, the highest rate of PO activity was obtained consistently at 5 min, and the activity remained at essentially the same level till 60 min; therefore, absorbance was recorded after 5 min. Value less than 2 units was considered negative [15]. Trypsin-activated HLS from Penaeus californiensis [11] was used as the proPO positive control. 2.10. Effect of trypsin, -chymotrypsin, laminarin, LPS, and SDS as elicitors All elicitors were purchased from Sigma. Trypsin (2 mg ml1), laminarin from Laminaria digitata (3 mg ml1), -chymotrypsin (2 mg ml1), LPS from Escherichia coli 055:B5 (3 µg ml1), and SDS (6 mg ml1) were diluted in pre-cooled Tris–HCl buffer (250 mM Tris; pH, 6.5). LHS (50 µl), JHS, plasma, or HLS samples were placed on a micro-well plate and incubated with 50 µl of the elicitor. Thereafter, 50 µl of 5 mM -DOPA was added as substrate. Optical density at 490 nm was recorded at 5, 10, 20, 30, and 60 min, using a micro-plate reader (Model 350, Bio–Rad). For the control, 50 µl of a sample was mixed with 50 µl of buffer and 50 µl of substrate. As blanks, 100 µl buffer was mixed with either 50 µl -DOPA to detect possible spontaneous oxidation of this substrate by light, or 50 µl of a sample to eliminate sample colour. 2.11. Effect of a protease inhibitor on proPO activation The effect of protease inhibitor cocktail P-2714 (Sigma, St Louis, MO) on laminarin-induced proPO activation in plasma and HLS in C. gigas, A. ventricosus, N. subnodosus, and A. maura was studied. In this experiment, 50 µl JHS, plasma, or HLS samples were pre-incubated with 25 µl of P-2714 at room temperature for 15 min. After that, 25 µl of 3 mg ml1 laminarin was added and further incubated at room temperature for 15 min. Three different controls were run simultaneously during the course of this experiment: JHS, plasma, or HLS samples were pre-incubated with Tris–HCl buffer as buffer control, laminarin, as positive control or P-2714 as negative control. Each 100-µl reaction volume was incubated with 50 µl of -DOPA, and PO activity was recorded. The amount of measurable proPO for laminarin, determined by subtracting PO activity in the buffer control from that of the positive control, was considered as 100%, and the level of proPO that remained in JHS, plasma, or HLS sample, after reaction with inhibitor and laminarin, was expressed as percent residual activity. 2.12. Protein determination Protein concentration of the studied samples was determined using the Bradford method [39], with bovine serum albumin (BSA) from Sigma as standard. The protein concentrations of mollusc samples analyzed were usually in the range of 0.12–3.50 mg ml1. Protein concentration in plasma and HLS of shrimp was 43.42 and 1.42 mg ml–1, respectively.
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Table 1 PO activity (units min1 mg protein1103) in plasma and haemocyte lysate of adults of four bivalve species, activated with trypsin, -chymotrypsin, SDS, laminarin, and LPS, and the effect of protease inhibitor cocktail P-2714 on proPO activation by laminarin Treatment
Control Trypsin -Chymotrypsin SDS Laminarin LPS P-2714 + Laminarin
Crassostrea gigas
Argopecten ventricosus
Nodipecten subnodosus
Atrina maura
HLS
Plasma
HLS
Plasma
HLS
Plasma
HLS
Plasma
4.52.3 9.82.7 9.33.9 14.62.3 9.63.8 8.60.5 52
7.35.4 18.7.0.5* 19.00.9* 22.12.9** 20.30.5** 19.20.5* 43
2.70.37 5.61.2 4.71.5 5.21.3 5.61.8 4.10.4 53
25.54.8 67.23.5** 55.52.5** 54.45.3** 50.57.9* 32.87.9 59
2.80.4 6.10.2** 4.40.5* 5.20.3** 7.00.6** 5.70.8** 54
18.50.6 32.23.2** 37.10.6** 40.72.2** 44.21.8** 42.82.2** 67
4.60.6 15.93.9 16.24.2 18.55.3 19.14.6 8.61.4 58
14.82.6 31.46.0* 25.93.7 27.43.7 33.73.9* 16.64.1 51
Values are the mean of four determinations, each one in quadruplicateSD. Values of inhibition by P-2714 are the mean of four determinations, each one in quadruplicate, and expressed as percent residual activity. * and ** denote significant differences as compared to control at P<0.05 and P<0.001, respectively.
2.13. Statistical analysis A one-way analysis of variance (ANOVA) using the F test was applied to analyse the differences among treatments and control and among species. P values lower than 0.05 were used to identify significant differences. Where significant ANOVA differences occurred among averages, Tukey’s HSD test was used to identify the nature of these differences at P<0.05. 3. Results 3.1. PO activity in larvae Control values for C. gigas, A. ventricosus, and N. subnodosus were 0.80.09, 0.70.13, 1.20.21, respectively. There was no PO activity either in the buffer control or after incubation of samples with proteolytic elicitors trypsin and -chymotrypsin, anionic detergent SDS, and microbial molecules laminarin and LPS. Large amounts of protein (2.18–3.5 mg ml1) were found in larval pools. 3.2. PO activity in juveniles PO activity was detected only in JHS of C. gigas and N. subnodosus. The activity obtained with the elicitors was not significantly different from the buffer control (3.90.59, 1.90.55, respectively). As in larvae, relatively large amounts of protein (0.88–1.06 mg ml1) were found in juvenile pool. 3.3. PO activity in HLS and plasma of adults Oxidation of -DOPA occurred in the buffer control of the four bivalve species (Table 1 ). Significant differences in the PO activity between buffer control and elicitors occurred in the plasma of all species, but in HLS of only N. subnodosus. The amounts of protein in HLS (0.23–1.4 mg ml1) and plasma (0.12–0.22 mg ml1) were lower than in larval and juvenile homogenates. 3.4. Effect of a protease inhibitor on proPO activation In the present study, the effect of P-2714 on the activation of proPO by laminarin was examined in the samples of C. gigas, A. ventricosus, N. subnodosus, and A. maura. Important reduction of PO activity was
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observed in HLS and plasma. The incubation of samples with P-2714, before reaction with 3 mg ml1 laminarin, consistently reduced PO activity up to 57% (Table 1). 4. Discussion In this study, PO activity was not detected in larval homogenates of C. gigas, A. ventricosus, and N. subnodosus. However, Dyrynda et al. [24] found PO activity in all disaggregated larval (trochophore and veliger) cells from M. edulis, using a cytochemical staining method. This discrepancy may be a consequence of the method used by Dyrynda et al. [24] and that employed here. In this study, we demonstrated that our method is reliable, since PO activity was found in two species of juveniles, which indicates the good resolution of the technique employed. Moreover, homogenates gave good results for determining lysozyme-like activity and other lysosomal enzymes in ripe eggs, larvae at different stages, and juveniles of C. gigas (Luna-Gonza´lez et al., unpublished). If the mollusc larvae lack a proPO system, these organisms, at this stage, would lack an important element of the defence system found in adults, partially explaining the high susceptibility of the bivalve larvae to bacterial infections [32–34,40–44]. Although detection of PO activity was positive in juvenile C. gigas and N. subnodosus, elicitors did not enhance PO activity and, therefore, the role of PO in juveniles of these species remains unclear. The lack of PO in A. ventricosus cannot be explained. Further work is needed to demonstrate the participation of other components of the defence system of adults in larvae, such as blood cells, antimicrobial proteins, lectins, lysosomal enzymes, superoxide anion, and peroxynitrite. Also to be explained is why PO was not activated by the elicitors tested, as it occurs in adults, and why PO activity was not detected in A. ventricosus juveniles. In adult molluscs, it is known that PO has an important role in shell growth and repair [23,45] and as part of the defence system [12]. In the present study, the spectrophotometric analysis revealed the oxidation of -DOPA by both HLS and plasma of adult C. gigas, A. ventricosus, N. subnodosus, and A. maura. Incubation of samples with elicitors enhanced PO activity in the samples of all species tested, suggesting the presence of a proPO system and its possible role in defence reactions, as was demonstrated by Asokan et al. [12] for the mussel P. viridis. In this mussel, PO activity was induced by trypsin as well as LPS and -1, 3-glucans. In other bivalve species, such as M. edulis, pre-incubation of the cells with 0.025% zymosansupernatant produced a ten-fold increase in the PO activity [3]. ProPO activity was also induced by trypsin in the gastropod mollusc Biomphalaria glabrata [46]. In many insects, crustaceans, and the bivalve P. viridis, the activation of proPO by non-self molecules has been shown to be susceptible to inhibition by serine protease inhibitors [9,12,47,48]. In this work, the incubation of plasma and HLS with protease inhibitor cocktail P-2714, prior to reaction with laminarin, prevented proPO activation up to 57%. The present study establishes that PO is present in juvenile C. gigas and N. subnodosus and its activity can be enhanced by the elicitors tested in adult specimens of the four species studied, suggesting that the proPO system plays a role as a defence mechanism.
Acknowledgements The authors are grateful to Cultivos del Mar Sudcaliforniano S. A. de C. V. and to Sol Azul S. A. de C. V. for providing broodstock and experimental organisms employed in this study. This work was supported by Consejo Nacional de Ciencia y Tecnologı´a, Mexico (CONACyT, project G-33953-B) and a PhD-student grant to A. Luna, and by CIBNOR project PAC-5. Thanks to Dr Jorge Herna´ndez Lo´pez for his critical review and comments on a previous version of this manuscript. CIBNOR editing staff improved the English language text.
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